Methamphetamine (Meth) abuse leads to behaviors that increase the risk of exposure to HIV1-3. NeuroAIDS, which affects >40% of HIV+ individuals, is a particularly severe complication of the infection causing neurological deficits, not decreased by antiviral drugs that do not cross the blood-brain-barrier efficiently 4. NeuroAIDS and peripheral pathologies associated to this co-morbidity have been reported to be aggravated by Meth5 6. Thus, the effects of Meth are predominant, but not restricted to the CNS. We have found that Meth modulates the phenotype of brain and peripheral innate immune cells in the SIV/Rhesus macaque model, increasing cytokine secretion and enriching virus targets CCR5+ cells7, and in vitro. We will investigate the hypothesis of direct action of Meth on innate immune cells, causing alterations that may enhance virus-triggered tissue-pathologies in HIV+ Meth abusers. In the present proposal, we will focus on innate immune cells existing in the brain and in the periphery, especially macrophages and microglia cells. Given that, we hypothesize that Meth can interfere with the anti-viral host immune response, by directly affecting innate immune cells of the macrophage/ microglia lineage. This can be an important component of increased neuropathology, particularly in neuroAIDS, since HIV brain targets are macrophages and microglia. We will determine if Meth modulates gene expression in CNS-derived immune cells in SIV-infected macaques, and changes sets of genes triggered by SIV in the brain by comparing gene expression in spleen and brain- cryopreserved immune cells (depleted of neurons and astro/oligoglia) in healthy and SIV-infected macaques, treated or not with Meth. Expression data will be compared using an integrative systems biology approach and validated. We will prioritize high score pathways, but favor molecules involved in cell cycle, oxidative stress, and inflammatory phenotype, in which the effects of Meth have been observed. Validation will be performed using qRTPCR and protein detection (western blot on brain and spleen frozen tissue fragments, FACS of frozen cell suspensions, and immunohistochemistry on paraffin-embedded tissue - already processed and available for all animals). In parallel, we will assess the ability of Meth to directly modulate macrophage/ microglia gene/protein expression and functional characteristics, using macrophage cell lines and human microglia primary cultures. Gene expression and functional assays assess the effects of Meth on cell cycle, oxidative stress, chemokine receptor and inflammatory molecule expression, mitochondrial functions and antigen processing/ presentation. This will allow the observation of direct effects on innate immune cells that get infected and predominate in the brain. These studies will help understand mechanisms regulating the severity of brain and other tissues pathology in HIV+ Meth users and reveal direct Meth targets on innate immune cells.